Project description: 30 lines Cytokinesis, the separation of a mother cell into two daughter cells, is one of life's most fundamental processes; it is central to the making of multicellular organisms and the transmission of genetic material across generations. The core machinery of cytokinesis is a contractile ring of actin, myosin motors and other proteins and is conserved from fungi to human. The contractile ring is connected to the inside of the cell cortex and its constriction leads the ingression of the plasma membrane into a furrow between the two cytoplasmic compartments that will become individual cells after the completion of cell division. Furrow ingression requires the cooperation between two main mechanisms: 1) constriction of actin filaments by the action of myosin motors and 2) the transmission of this contractile force to the plasma membrane via anchor proteins that connect the actomyosin bundle to the plasma membrane. We found that the proteins of the contractile ring occupy distinct layers with plasma membrane-interacting proteins in the outer layer adjacent to the cortex and the force- producing myosin motors in the inner layer. The next frontier in understanding the mechanism of cytokinesis is to determine how these proteins are organized into complex structures, how these structures move within the contractile ring and are removed from the ring during constriction, and how this dynamic architecture governs the force-generation function of the contractile rings. The objective of this application is to determine the anchoring role of the outer ring and the tension-force producing role of the inner ring by uncovering their dynamic architecture and effects on mechanics of constriction. Our hypothesis is that furrow ingression results from contractile forces produced in the inner layer of the contractile ring, conveyed to the plasma membrane via anchoring achieved by proteins in the outer layer. We plan to test our central hypothesis with the following Specific Aims: 1) determine how cytokinetic node proteins anchor the contractile ring and transmit contractile forces to the plasma membrane during furrow ingression and 2) determine how the molecular architecture of the inner ring governs the constriction of the contractile ring during furrow ingression. The proposed research in this application is innovative because we will use a unique combination of high-speed Fluorescence Photoactivation Localization Microscopy (hsFPALM) in live cells to determine protein organization and its dynamics with laser microsurgery to probe the mechanics of this tension-force producing machine in live cells. The proposed research in this application is significant as it will result in the identification of previously unknown parameters for new molecular and functional models of the contractile ring. As the proteins of the contractile ring are conserved from yeast to human, we expect that the core mechanism of cytokinesis elucidated in fission yeast will act as a template for understanding the mechanism of cytokinesis and even perhaps other non-musclecontractile cellular machines in other species.